Fungamyl-like alpha-amylase variants

ABSTRACT

The invention relates to a variant of a parent Fungamyl-like fungal alpha-amylase, which exhibits improved thermal stability at acidic pH suitable for, e.g., starch processes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 10/820,200filed Apr. 7, 2004, now U.S. Pat. No. 7,160,710, which is a continuationof U.S. application Ser. No. 09/710,339, filed Nov. 09, 2000, now U.S.Pat. No. 7,005,288, which claims, under 35 U.S.C. 119, the benefit ofU.S. provisional application Ser. No. 60/165,786, filed Nov. 16, 1999,and priority from Danish application no. PA 1999 01617 filed Nov. 10,1999, the contents of which are fully incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to alpha-amylase variants (mutants) ofFungamyl™-like alpha-amylases, in particular with improved thermalstability at acidic pH. The invention also relates to the use of suchvariants.

BACKGROUND OF THE INVENTION

Alpha-Amylases (alpha-1,4-glucan-4-glucanohydrolases, EC. 3.2.1.1)constitute a group of enzymes which catalyze hydrolysis of starch andother linear and branched 1,4-glucosidic oligo- and polysaccharides.

There is a very extensive body of patent and scientific literaturerelating to this industrially very important class of enzymes. A numberof alpha-amylase referred to as “Termamyl®-like alpha-amylases” andvariants thereof are known from, e.g., WO 90/11352, WO 95/10603, WO95/26397, WO 96/23873 and WO 96/23874. Termamyl-like alpha-amylases arevery thermostable and therefore suitable for processes carried out athigh temperatures such as starch liquefaction in dextrose productionprocesses.

Another group of alpha-amylases are referred to as “Fungamyl®-likealpha-amylases”, which are alpha-amylases related to the alpha-amylasederived from Aspergillus oryzae (and shown in SEQ ID NO: 1). TheseFungamyl-like alpha-amylases have a relatively low thermostability (thecommercial product sold under the tradename FUNGAMYL™ by Novo Nordisk,Denmark, has a optimum around 55° C.) and is therefore not suitable forprocesses carried out at high temperatures. Fungamyl-like alpha-amylasesare today used for making syrups for, e.g., the brewing industry. Suchprocesses are operated at around 60° C. resulting in that usually in therange of double the enzyme dosage must be used to compensate for the lowthermostability. Further, at 55° C. infection problems may occur.

As such processes today furthermore are carried out at a pH of 5.5,instead of, e.g., pH 4.5, pH adjustment and addition of Sodium to thesyrups are necessitated.

Therefore, it would be advantageous to provide a Fungamyl-likealpha-amylase with increased thermostability preferably at an acidic pH.

BRIEF DISCLOSURE OF THE INVENTION

The object of the present invention is to provide Fungamyl-likealpha-amylase variant, in particular with improved thermostablilityespecially at acidic pH.

The term “an alpha-amylase variant with improved thermostability” meansin the context of the present invention an alpha-amylase variant, whichhas a higher thermostability than corresponding parent alpha-amylases.The determination of thermostability is described below in the Materialsand Method section.

The inventors have provided improved Fungamyl-like alpha-amylasevariants as will be described further below.

DETAILED DISCLOSURE OF THE INVENTION

A goal of the work underlying the present invention was to improve thethermal stability, in particular at acidic pH of Fungamyl-likealpha-amylases.

Identifying Positions and/or Regions to be Mutated to Obtain ImprovedThermostability

Molecular dynamics (MD) simulations indicate the mobility of the aminoacids in the protein structure (see McCammon, J A and Harvey, S C.,(1987), “Dynamics of proteins and nucleic acids”. Cambridge UniversityPress.). Such protein dynamics are often compared to thecrystallographic B-factors (see Stout, G H and Jensen, L H, (1989),“X-ray structure determination”, Wiley) or the B-factors themselves. Byrunning the MD simulation at different protonation states of thetitrateable residues, the pH related mobility of residues are simulated.Regions having the highest mobility or flexibility (here isotropicfluctuations) are selected for random mutagenesis. It is here understoodthat the high mobility found in certain areas of the protein, can bethermally improved by substituting residues in these residues. Thesubstitutions are directed against residues that have bigger side-chainsand/or which have capability of forming improved contacts to residues inthe near environment. The parent Fungamyl® alpha-amylase backbone shownin SEQ ID NO: 2 derived from Aspergillus oryzae was used for the MDsimulation.

Regions found by Molecular dynamics (MD) simulations or B factorexamination (as enclosed to the Protein Data Base (PDB)(www.rcsb.org)file 6TAA (Swift, H. J., Brady, L., Derewenda, Z. S., Dodson, E. J.,Dodson, G. G., Turkenburg, J. P., Wilkinson, A. J.: Structure andmolecular model refinement of Aspergillus oryzae (TAKA) alpha-amylase:an application of the simulated-annealing method. Acta Crystallogr B 47pp. 535 (1991)) to be suitable for mutation when wanting to obtain, inparticular increased thermal stability are the following:

-   Region 98-110,-   Region 150-160,-   Region 161-167,-   Region 280-288,-   Region 448-455,-   Region 468-475.

The above regions are show to be flexible. Making said regions morerigid would make the molecule more thermostable.

Accordingly, in a first aspect the present invention relates to avariant of a parent Fungamyl-like alpha-amylase comprising one or moremutations in the regions and positions described further below.

Nomenclature

In the present description and claims, the conventional one-letter andthree-letter codes for amino acid residues are used. For ease ofreference, alpha-amylase variants of the invention are described by useof the following nomenclature:

Original amino acid(s):position(s):substituted amino acid(s)

According to this nomenclature, for instance the substitution of alaninefor asparagine in position 30 is shown as:

Ala30Asn or A30N

a deletion of alanine in the same position is shown as:

Ala30* or A30*

and insertion of an additional amino acid residue, such as lysine, isshown as:

Ala30AlaLys or A30AK

A deletion of a consecutive stretch of amino acid residues, such asamino acid residues 30-33, is indicated as (30-33)* or Δ(A30-N33).

Where a specific alpha-amylase contains a “deletion” in comparison withother alpha-amylases and an insertion is made in such a position this isindicated as:

*36Asp or *36D

for insertion of an aspartic acid in position 36

Multiple mutations are separated by plus signs, i.e.:

Ala30Asp +Glu34Ser or A30N+E34S

representing mutations in positions 30 and 34 substituting alanine andglutamic acid for asparagine and serine, respectively. Multiplemutations may also be separated as follows, i.e., meaning the same asthe plus sign:

Ala30Asp/Glu34Ser or A30N/E34S

When one or more alternative amino acid residues may be inserted in agiven position it is indicated as

A30N,E or

A30N or A30E

Furthermore, when a position suitable for modification is identifiedherein without any specific modification being suggested, it is to beunderstood that any amino acid residue may be substituted for the aminoacid residue present in the position.

Thus, for instance, when a modification of an alanine (A) in position 30is mentioned, but not specified, or specified as “A30X”, it is to beunderstood that the alanine may be deleted or substituted for any otheramino acid, i.e., any one of: R,N,D,A,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V.

Fungamyl-Like Alpha-Amylases

Parent Fungamyl-like alpha-amylase are according to the presentinvention enzymes with alpha-amylase activity which either have at least60%, preferably at least 70%, more preferably at least 80%, even morepreferably at least 90%, even more preferably at least 93%, even morepreferably at least 95%, even more preferably at least 97%, even morepreferably at least 99% identity to the DNA sequence shown in SEQ ID NO:1 encoding the alpha-amylase and/or the mature part of the alpha-amylaseprotein sequence shown in SEQ ID NO: 2 and/or structurally resembles thethree-dimensional structure of the FUNGAMYL® alpha-amylase shown in SEQID NOS: 1 and 2, and further disclosed in the Protein Data Base (PDB)(www.rcsb.org) file 6TAA (Swift, H. J., Brady, L., Derewenda, Z. S.,Dodson, E. J., Dodson, G. G., Turkenburg, J. P., Wilkinson, A. J.:Structure and molecular model refinement of Aspergillus oryzae (TAKA)alpha-amylase: an application of the simulated-annealing method. ActaCrystallogr B 47 pp. 535 (1991) and/or is encoded by a DNA sequence,which hybridizes to the part of the DNA sequence shown in SEQ ID NO: 1encoding the mature part of the alpha-amylase shown in SEQ ID NO: 2 ofthe present specification.

Specific examples of such alpha-amylases covered by the definition“Fungamyl-like alpha-amylases” include the Aspergillus oryzae TAKAalpha-amylase (EP 238 023) and shown in SEQ ID NO: 2, and the A. nigeralpha-amylase disclosed in EP 383,779 B2 (section (see also the cloningof the A. niger gene in Example 1).

In an embodiment the Fungamyl-like alpha-amylase is derived from afungal organism, in particular of the genus Aspergillus, in particular Aoryzae or A. niger.

Commercially Available Parent Fungamyl-Like Alpha-Amylases

Commercially available parent Fungamyl-like alpha-amylases includeFungamyl® (from Novo Nordisk, Denmark). Fungamyl® is a fungalalpha-amylase obtained from a selected strain of Aspergillus oryzae. Inthe starch industry, Fungamyl® is used for production of high maltosesyrups, 45-60% maltose (2-7% glucose) or high conversion syrups, DE60-70, 35-43% glucose, 30-37% maltose. Other commercial fungalalpha-amylases include Clarase™ (from Genencor Int., USA) derived fromAspergillus oryzae; and Maltamyl™ (from Enzyme Biosystems) derived fromAspergillus niger.

In the brewing industry, FUNGAMYL® (and similar products) is addedduring fermentation in order to increase fermentability of the wort.

In the alcohol industry, FUNGAMYL® may be used for liquefaction ofstarch in a distillery mash if the existing equipment favorslow-temperature liquefaction (55-60° C.). FUNGAMYL® (and similarproducts) is also used for baking and can be used for all types of breadand baked products. For instance FUNGAMYL® improves the dough stability,result in greater bread volume, improves crumb softness and give thecrust a darker color.

Alpha-Amylase Variants of the Invention

In the first aspect the invention relates to a variant of a parentFungamyl-like alpha-amylase comprising one or more mutation(s) in thefollowing positions(s) or region(s) in the amino acid sequence shown inNO: 2:

-   Region 98-110,-   Region 150-160,-   Region 161-167;-   Region 280-288,-   Region 448-455,-   Region 468-475, and/or in a corresponding position or region in a    homologous Fungamyl-like alpha-amylase which displays at least 60%    identity with the amino acid sequences shown in SEQ ID NO: 2. In an    embodiment the region mutated is Region 98-110.

In an embodiment the region mutated is Region 98-110, more specificallyone or more of the following positions: 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110.

Specific substitutions are

-   98X, preferably T;-   99X, preferably T;-   100F,Y,W,I,M; preferably Y;-   101R;-   102S,T,V;-   103I,F,V, preferably I;-   104T,V,I;-   105X, preferably A;-   106V,L,N,D,Q,E, preferably V;-   107V,I,M;-   108Y,R,K;-   109D,N,Q;-   110Q.

In an embodiment the region mutated is Region 150-160, more specificallyone or more of the following positions:

-   150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160.    Specific substitutions are-   151Y,Q,L,I,R, preferably Y;-   152V,L;-   153T,N,S, preferably S;-   154L,Y,V,T,S, preferably L;-   155F,N,L;-   156X, preferably D,N,S,T;-   157S,T,N;-   158E,Y-   159X, preferably S,A;-   160X, preferably N;

In an embodiment the region mutated is Region 161-167, more specificallyone or more of the following positions: 161, 162, 163, 164, 165, 166,167.

Specific substitutions are

-   161I,S,T;-   162D,N,Q,Y;-   163E,Q,N;-   166V,F,Y,I,S,T,prefeably V,F,Y;-   167A;

In an embodiment the region mutated is Region 280-288, more specificallyone or more of the following positions:

-   280, 281, 282, 283, 284, 285, 286, 287, 288.    Specific substitutions are-   280Q,Y,R;-   281X, preferably T,A;-   282X, preferably S,T;-   285L,N;-   286X, preferably D;-   287V,S,A;-   288N,F,Y,E,D, preferably N;

In an embodiment the region mutated is Region 448-455 more specificallyone or more of the following positions:

-   448, 449, 450, 451, 452, 453, 454, 455.    Specific substitutions are:-   448X, preferably A,L,Y,S,T;-   449x, preferably L,V,S,T;-   450I,T,L;-   452I,L;-   454I,L;-   455D,E,S,T.

In an embodiment the region mutated is Region 468-475 more specificallyone or more of the following positions:

-   468, 469, 470, 471, 472, 473, 474, 475.    Specific substitutions are:-   468F,Y,H;-   469E,D,Q,N;-   470X, preferably A,S,T;-   471N,T,K,R,F,Y preferably N,T,Y;-   472R;-   473L,N,Y;-   475X, preferably T,R.    Improved Stability at Acidic pH

One object of the invention is to make the Fungamyl-like alpha-amylasemore acidic in comparison to the parent alpha-amylase (i.e.,corresponding un-mutated alpha-amylase).

That a Fungamyl-like alpha-amylase variant is more acidic than theparent Fungamyl-like alpha-amylase means that the stability at acidic pHis higher that for the corresponding parent alpha-amylase. That theamylase is more acidic may be determined as described in the “Materials& Methods” section.

The term “acidic pH” means at least in the context of the presentinvention a pH in the range from 4-6, such as 4-5, in particular4.2-4.7.

Providing more acidic fungal alpha-amylases are desired, because itopens up for the possibility of using the fungal alpha-amylase varianttogether with or simultaneously with a suitable glucoamylase, e.g.,during the (dextrinazation) saccharification step in starch processes.

Thermal Stability

One object of the invention is to provide a more thermostableFungamyl-like alpha-amylase.

That a Fungamyl-like alpha-amylase variant is more thermostable than theparent Fungamyl-like alpha-amylase means that the temperature optimumhas been pushed towards a higher temperature. That the amylase is morethermostable may be determined as described in the “Materials & Methods”section.

Providing more thermostable fungal alpha-amylases is desired because itrenders a more efficient and/or faster liquefaction step possible.Further, the liquefaction temperature is less sensitive and may even beincreased (i.e., less cooling necessary. Further, the risk of infectionis also reduced.

It is to be understood that variants of the invention may have both amore stable at acidic pH and be more thermostable, in particular atacidic pH.

Homology (Identity)

The homology (identity) referred to above of the parent alpha-amylase isdetermined as the degree of identity between two protein or DNAsequences indicating a derivation of the first sequence from the second.The homology (identity) may suitably be determined by means of computerprograms known in the art such as GAP provided in the GCG programpackage (Program Manual for the Wisconsin Package, Version 8, August1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of MolecularBiology, 48, p. 443-453). The (default) GAP creation penalty is 5.0 andthe GAP extension penalty of 0.3, respectively, for nucleic acidicsequence comparison; and (default) GAP creation penalty is 3.0 and GAPextension penalty of 0.1, respectively, for protein sequence comparison.GAP uses the method of Needleman and Wunsch, (1970), J. Mol. Biol. 48,p.443-453, to make alignments and to calculate the identity.

Using GAP with the above settings for polypeptide or DNA sequencecomparison a parent Fungamyl-like alpha-amylase has a degree of identitypreferably of at least 60%, such as 70%, at least 80%, at least 90%,more preferably at least 95%, more preferably at least 97%, and mostpreferably at least 99% with the mature part of the amino acid sequenceshown in SEQ ID NO: 2 and encoding part of the DNA sequence shown in SEQID NO: 1.

In a preferred embodiment the variant of the invention has improvedthermal stability, in particular at acidic pH.

Hybridisation

Oligonucleotide probes used in the characterisation of the Fungamyl-likealpha-amylase may suitably be prepared on the basis of the full orpartial nucleotide or amino acid sequence of the alpha-amylase inquestion.

Suitable conditions for testing hybridisation involve pre-soaking in5×SSC and prehybridizing for 1 hour at ˜40° C. in a solution of 20%formamide, 5× Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and50 mg of denatured sonicated calf thymus DNA, followed by hybridisationin the same solution supplemented with 100 mM ATP for 18 hours at 40°C., followed by three times washing of the filter in 2×SSC, 0.2% SDS at40° C. for 30 minutes (low stringency), preferred at 50° C. (mediumstringency), more preferably at 65° C. (high stringency), even morepreferably at 75° C. (very high stringency). More details about thehybridisation method can be found in Sambrook et al., Molecular_Cloning:A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989.

In the present context, “derived from” is intended not only to indicatean alpha-amylase produced or producible by a strain of the organism inquestion, but also an alpha-amylase encoded by a DNA sequence isolatedfrom such strain and produced in a host organism transformed with saidDNA sequence. Finally, the term is intended to indicate analpha-amylase, which is encoded by a DNA sequence of synthetic and/orcDNA origin and which has the identifying characteristics of thealpha-amylase in question. The term is also intended to indicate thatthe parent alpha-amylase may be a variant of a naturally occurringalpha-amylase, i.e., a variant, which is the result of a modification(insertion, substitution, deletion) of one or more amino acid residuesof the naturally occurring alpha-amylase.

Cloning a DNA sequence encoding an Fungamyl-like alpha-amylaseCloning aDNA sequence encoding an a-amylaseCloning a DNA sequence encoding ana-amylaseCloning a DNA sequence encoding an a-amylaseCloning a DNAsequence encoding an a-amylaseCloning a DNA sequence encoding ana-amylaseCloning a DNA sequence encoding an a-amylaseCloning a DNAsequence encoding an a-amylase The DNA sequence encoding a parentFungamyl-like alpha-amylase may be isolated from any cell ormicroorganism producing alpha-amylases, using various methods well knownin the art. First, a genomic DNA and/or cDNA library should beconstructed using chromosomal DNA or messenger RNA from the organismthat produces the alpha-amylase to be studied. Then, if the amino acidsequence of the alpha-amylase is known, labeled oligonucleotide probesmay be synthesized and used to identify alpha-amylase-encoding clonesfrom a genomic library prepared from the organism in question.Alternatively, a labelled oligonucleotide probe containing sequenceshomologous to another known alpha-amylase gene could be used as a probeto identify alpha-amylase-encoding clones, using hybridization andwashing conditions of lower stringency.

Yet another method for identifying alpha-amylase-encoding clones wouldinvolve inserting fragments of genomic DNA into an expression vector,such as a plasmid, transforming alpha-amylase-negative bacteria with theresulting genomic DNA library, and then plating the transformed bacteriaonto agar containing a substrate for alpha-amylase (i.e., maltose),thereby allowing clones expressing the alpha-amylase to be identified.

Alternatively, the DNA sequence encoding the enzyme may be preparedsynthetically by established standard methods, e.g. the phosphoroamiditemethod described S. L. Beaucage and M. H. Caruthers, (1981), TetrahedronLetters 22, p. 1859-1869, or the method described by Matthes et al.,(1984), EMBO J. 3, p. 801-805. In the phosphoroamidite method,oligonucleotides are synthesized, e.g., in an automatic DNA synthesizer,purified, annealed, ligated and cloned in appropriate vectors.

Finally, the DNA sequence may be of mixed genomic and synthetic origin,mixed synthetic and CDNA origin or mixed genomic and CDNA origin,prepared by ligating fragments of synthetic, genomic or cDNA origin (asappropriate, the fragments corresponding to various parts of the entireDNA sequence), in accordance with standard techniques. The DNA sequencemay also be prepared by polymerase chain reaction (PCR) using specificprimers, for instance as described in U.S. Pat. No. 4,683,202 or R. K.Saiki et al., (1988), Science 239, pp. 487-491.

Site-Directed Mutagenesis

Once a Fungamyl-like alpha-amylase-encoding DNA sequence has beenisolated, and desirable sites for mutation identified, mutations may beintroduced using synthetic oligonucleotides. These oligonucleotidescontain nucleotide sequences flanking the desired mutation sites. In aspecific method, a single-stranded gap of DNA, thealpha-amylase-encoding sequence, is created in a vector carrying thealpha-amylase gene. Then the synthetic nucleotide, bearing the desiredmutation, is annealed to a homologous portion of the single-strandedDNA. The remaining gap is then filled in with DNA polymerase I (Klenowfragment) and the construct is ligated using T4 ligase. A specificexample of this method is described in Morinaga et al., (1984),Biotechnology 2, p. 646-639. U.S. Pat. No. 4,760,025 disclose theintroduction of oligonucleotides encoding multiple mutations byperforming minor alterations of the cassette. However, an even greatervariety of mutations can be introduced at any one time by the Morinagamethod, because a multitude of oligonucleotides, of various lengths, canbe introduced.

Another method for introducing mutations into α-amylase-encoding DNAsequences is described in Nelson and Long, (1989), AnalyticalBiochemistry 180, p. 147-151. It involves the 3-step generation of a PCRfragment containing the desired mutation introduced by using achemically synthesized DNA strand as one of the primers in the PCRreactions. From the PCR-generated fragment, a DNA fragment carrying themutation may be isolated by cleavage with restriction endonucleases andreinserted into an expression plasmid.

Random Mutagenesis

Random mutagenesis is suitably performed either as localized orregion-specific random mutagenesis in at least three parts of the genetranslating to the amino acid sequence shown in question, or within thewhole gene.

The random mutagenesis of a DNA sequence encoding a parent glucoamylasemay be conveniently performed by use of any method known in the art.

In relation to the above, a further aspect of the present inventionrelates to a method for generating a variant of a parent Fungamyl-likealpha-amylase, wherein the variant exhibits increased thermal stability,especially at acidic pH, relative to the parent, the method comprising:

-   -   (a) subjecting a DNA sequence encoding the parent Fungamyl-like        alpha-amylase to random mutagenesis,    -   (b) expressing the mutated DNA sequence obtained in step (a) in        a host cell, and    -   (c) screening for host cells expressing an alpha-amylase variant        which has an altered property (i.e., thermal stability) relative        to the parent Fungamyl-like alpha-amylase.

Step (a) of the above method of the invention is preferably performedusing doped primers, as described in the working examples herein (videinfra).

For instance, the random mutagenesis may be performed by use of asuitable physical or chemical mutagenizing agent, by use of a suitableoligonucleotide, or by subjecting the DNA sequence to PCR generatedmutagenesis. Furthermore, the random mutagenesis may be performed by useof any combination of these mutagenizing agents. The mutagenizing agentmay, e.g., be one, which induces transitions, transversions, inversions,scrambling, deletions, and/or insertions.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) ir-radiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues. When such agents are used, themutagenesis is typically performed by incubating the DNA sequenceencoding the parent enzyme to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions for themutagenesis to take place, and selecting for mutated DNA having thedesired properties.

When the mutagenesis is performed by the use of an oligonucleotide, theoligonucleotide may be doped or spiked with the three non-parentnucleotides during the synthesis of the oligonucleotide at thepositions, which are to be changed. The doping or spiking may be done sothat codons for unwanted amino acids are avoided. The doped or spikedoligonucleotide can be incorporated into the DNA encoding theglucoamylase enzyme by any published technique, using, e.g., PCR, LCR orany DNA polymerase and ligase as deemed appropriate.

Preferably, the doping is carried out using “constant random doping”, inwhich the percentage of wild type and mutation in each position ispredefined. Furthermore, the doping may be directed toward a preferencefor the introduction of certain nucleotides, and thereby a preferencefor the introduction of one or more specific amino acid residues. Thedoping may be made, e.g., so as to allow for the introduction of 90%wild type and 10% mutations in each position. An additionalconsideration in the choice of a doping scheme is based on genetic aswell as protein-structural constraints. The doping scheme may be made byusing the DOPE program which, inter alia, ensures that introduction ofstop codons is avoided.

When PCR-generated mutagenesis is used, either a chemically treated ornon-treated gene encoding a parent glucoamylase is subjected to PCRunder conditions that increase the mis-incorporation of nucleotides(Deshler 1992; Leung et al., Technique, Vol.1, 1989, pp. 11-15).

A mutator strain of E. coli (Fowler et al., Molec. Gen. Genet., 133,1974, pp. 179-191), S. cereviseae or any other microbial organism may beused for the random mutagenesis of the DNA encoding the glucoamylase by,e.g., transforming a plasmid containing the parent glycosylase into themutator strain, growing the mutator strain with the plasmid andisolating the mutated plasmid from the mutator strain. The mu-ta-tedplasmid may be subsequently transformed into the expression organism.

The DNA sequence to be mutagenized may be conveniently present in agenomic or cDNA library prepared from an organism expressing the parentglucoamylase. Alternatively, the DNA sequence may be present on asuitable vector such as a plasmid or a bacteriophage, which as such maybe incubated with or otherwise exposed to the mutagenising agent. TheDNA to be mutagenized may also be present in a host cell either by beingintegrated in the genome of said cell or by being present on a vectorharboured in the cell. Finally, the DNA to be mutagenized may be inisolated form. It will be understood that the DNA sequence to besubjected to random mutagenesis is preferably a cDNA or a genomic DNAsequence.

In some cases it may be convenient to amplify the mutated DNA sequenceprior to performing the expression step b) or the screening step c).Such amplification may be performed in accordance with methods known inthe art, the presently preferred method being PCR-generatedamplification using oligonucleotide primers prepared on the basis of theDNA or amino acid sequence of the parent enzyme.

Subsequent to the incubation with or exposure to the mutagenising agent,the mutated DNA is expressed by culturing a suitable host cell carryingthe DNA sequence under conditions allowing expression to take place. Thehost cell used for this purpose may be one which has been transformedwith the mutated DNA sequence, optionally present on a vector, or onewhich was carried the DNA sequence encoding the parent enzyme during themutagenesis treatment. Examples of suitable host cells are thefollowing: gram positive bacteria such as Bacillus subtilis, Bacilluslicheniformis, Bacillus lentus, Bacillus brevis, Bacillusstearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens,Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillusmegaterium, Bacillus thuringiensis, Streptomyces lividans orStreptomyces murinus; and gram-negative bacteria such as E. coli.

The mutated DNA sequence may further comprise a DNA sequence encodingfunctions permitting expression of the mutated DNA sequence.

Localized Random Mutagenesis

The random mutagenesis may be advantageously localized to a part of theparent alpha-amylase in question. This may, e.g., be advantageous whencertain regions of the enzyme have been identified to be of particularimportance for a given property of the enzyme, and when modified areexpected to result in a variant having improved properties. Such regionsmay normally be identified when the tertiary structure of the parentenzyme has been elucidated and related to the function of the enzyme.

The localized, or region-specific, random mutagenesis is convenientlyperformed by use of PCR generated mutagenesis techniques as describedabove or any other suitable technique known in the art. Alternatively,the DNA sequence encoding the part of the DNA sequence to be modifiedmay be isolated, e.g., by insertion into a suitable vector, and saidpart may be subsequently subjected to mutagenesis by use of any of themutagenesis methods discussed above.

Alternative methods for providing variants of the invention include geneshuffling, e.g., as described in WO 95/22625 (from Affymax TechnologiesN.V.) or in WO 96/00343 (from Novo Nordisk A/S), or other correspondingtechniques resulting in a hybrid enzyme comprising the mutation(s),e.g., substitution(s) and/or deletion, in question.

Expression of Alpha-Amylase Variants

According to the invention, a DNA sequence encoding the variant producedby methods described above, or by any alternative methods known in theart, can be expressed, in enzyme form, using an expression vector whichtypically includes control sequences encoding a promoter, operator,ribosome binding site, translation initiation signal, and, optionally, arepressor gene or various activator genes.

Expression Vector

The recombinant expression vector carrying the DNA sequence encoding analpha-amylase variant of the invention may be any vector which mayconveniently be subjected to recombinant DNA procedures, and the choiceof vector will often depend on the host cell into which it is to beintroduced. The vector may be one which, when introduced into a hostcell, is integrated into the host cell genome and replicated togetherwith the chromosome(s) into which it has been integrated. Examples ofsuitable expression vectors include pMT838.

Promoter

In the vector, the DNA sequence should be operably connected to asuitable promoter sequence. The promoter may be any DNA sequence, whichshows transcriptional activity in the host cell of choice and may bederived from genes encoding proteins either homologous or heterologousto the host cell.

Examples of suitable promoters for directing the transcription of theDNA sequence encoding an alpha-amylase variant of the invention,especially in a bacterial host, are the promoter of the lac operon ofE.coli, the Streptomyces coelicolor agarase gene dagA promoters, thepromoters of the Bacillus licheniformis alpha-amylase gene (amyL), thepromoters of the Bacillus stearothermophilus maltogenic amylase gene(amyM), the promoters of the Bacillus amyloliquefaciens alpha-amylase(amyQ), the promoters of the Bacillus subtilis xylA and xylB genes etc.For transcription in a fungal host, examples of useful promoters arethose derived from the gene encoding A. oryzae TAKA amylase, the TPI(triose phosphate isomerase) promoter from S. cerevisiae (Alber et al.(1982), J. Mol. Appl. Genet 1, p. 419-434, Rhizo-mucor miehei asparticproteinase, A. niger neutral alpha-amylase, A. niger acid stablealpha-amylase, A. niger glucoamylase, Rhizo-mucor miehei lipase, A.oryzaealkaline protease, A. oryzaetriose phosphate isomerase or A.niulans acetamidase.

Expression Vector

The expression vector of the invention may also comprise a suitabletranscription terminator and, in eukaryotes, poly-adenylation sequencesoperably connected to the DNA sequence encoding the alpha-amylasevariant of the invention. Termination and polyadenylation sequences maysuitably be derived from the same sources as the promoter.

The vector may further comprise a DNA sequence enabling the vector toreplicate in the host cell in question. Examples of such sequences arethe origins of replication of plasmids pUC19, pACYC177, pUB110, pE194,pAMB1 and pIJ702.

The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell, such as the dalgenes from B. subtilis or B. licheniformis, or one which confersantibiotic resistance such as ampicillin, kanamycin, chloramphenicol ortetracyclin resistance. Furthermore, the vector may comprise Aspergillusselection markers such as amdS, argB, niaD and sC, a marker giving riseto hygromycin resistance, or the selection may be accomplished byco-transformation, e.g., as described in WO 91/17243.

The procedures used to ligate the DNA construct of the inventionencoding a glucoamylase variant, the promoter, terminator and otherelements, respectively, and to insert them into suitable vectorscontaining the information necessary for replication, are well known topersons skilled in the art (cf., for instance, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor,1989).

Host Cells

The cell of the invention, either comprising a DNA construct or anexpression vector of the invention as defined above, is advantageouslyused as a host cell in the recombinant production of an alpha-amylasevariant of the invention. The cell may be transformed with the DNAconstruct of the invention encoding the variant, conveniently byintegrating the DNA construct (in one or more copies) in the hostchromosome. This integration is generally considered to be an advantageas the DNA sequence is more likely to be stably maintained in the cell.Integration of the DNA constructs into the host chromosome may beperformed according to conventional methods, e.g., by homologous orheterologous recombination. Alternatively, the cell may be transformedwith an expression vector as described above in connection with thedifferent types of host cells.

The cell of the invention may be a cell of a higher organism such as amammal or an insect, but is preferably a microbial cell, e.g., abacterial or a fungal (including yeast) cell.

Examples of suitable bacteria are Gram-positive bacteria such asBacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillusbrevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacilluslautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomyceslividans or Streptomyces murinus, or gram-negative bacteria such asE.coli. The transformation of the bacteria may, for instance, beeffected by protoplast transformation or by using competent cells in amanner known per se.

The yeast organism may favorably be selected from a species ofSaccharomyces or Schizosaccharomyces, e.g., Saccharomyces cerevisiae.

The host cell may also be a filamentous fungus, e.g., a strain belongingto a species of Aspergillus, most preferably Aspergillus oryzae orAspergillus niger, or a strain of Fusarium, such as a strain of Fusariumoxysporium, Fusarium graminearum (in the perfect state named Gribberellazeae, previously Sphaeria zeae, synonym with Gibberella roseum andGibberella roseum f. sp. cerealis), or Fusarium sulphureum (in theprefect state named Gibberella puricaris, synonym with Fusariumtrichothecioides, Fusarium bactridioides, Fusarium sambucium, Fusariumroseum, and Fusarium roseum var. graminearum), Fusarium cerealis(synonym with Fusarium crokkwellnse), or Fusarium venenatum.

In a preferred embodiment of the invention the host cell is a proteasedeficient or protease minus strain. This may for instance be theprotease deficient strain of the genus Aspergillus, in particular astrain of A. oryzae, such as A. oryzae JaL125 having the alkalineprotease gene named “alp” deleted. This strain is described in WO97/35956 (Novo Nordisk).

Filamentous fungi cells may be transformed by a process involvingprotoplast formation and transformation of the protoplasts followed byregeneration of the cell wall in a manner known per se. The use ofAspergillus as a host micro-organism is described in EP 238 023 (NovoNordisk), the contents of which are hereby incorporated by reference.

Method of Producing an Alpha-Amylase Variant of the Invention

In a yet further aspect, the present invention relates to a method ofproducing an alpha-amylase variant of the invention, which methodcomprises cultivating a host cell under conditions conducive to theproduction of the variant and recovering the variant from the cellsand/or culture medium.

The medium used to cultivate the cells may be any conventional mediumsuitable for growing the host cell in question and obtaining expressionof the alpha-amylase variant of the invention. Suitable media areavailable from commercial suppliers or may be prepared according topublished recipes (e.g., as described in catalogues of the American TypeCulture Collection).

The alpha-amylase variant secreted from the host cells may convenientlybe recovered from the culture medium by well-known procedures, includingseparating the cells from the medium by centrifugation or filtration,and precipitating proteinaceous components of the medium by means of asalt such as ammonium sulphate, followed by the use of chromatographicprocedures such as ion exchange chromatography, affinity chromatography,or the like.

Starch Conversion

The present invention provides a method of using alpha-amylase variantsof the invention for producing glucose or maltose or the like fromstarch.

Generally, the method includes the steps of partially hydrolyzingprecursor starch in the presence of alpha-amylase and then furtherhydrolyzing the release of D-glucose from the non-reducing ends of thestarch or related oligo- and polysaccharide molecules in the presence ofglucoamylase by cleaving alpha-(1

4) and alpha-(1

6) glucosidic bonds.

The partial hydrolysis of the precursor starch utilizing alpha-amylaseprovides an initial breakdown of the starch molecules by hydrolyzinginternal alpha-(1

4)-linkages. In commercial applications, the initial hydrolysis usingalpha-amylase is run at a temperature of approximately 105° C. A veryhigh starch concentration is processed, usually 30% to 40% solids. Theinitial hydrolysis is usually carried out for five minutes at thiselevated temperature. The partially hydrolyzed starch can then betransferred to a second tank and incubated for approximately one hour ata temperature of 85° to 90° C. to derive a dextrose equivalent (D.E.) of10 to 15.

The step of further hydrolyzing the release of D-glucose from thenon-reducing ends of the starch or related oligo- and polysaccharidesmolecules in the presence of glucoamylase is normally carried out in aseparate tank at a reduced temperature between 30° and 60° C. Preferablythe temperature of the substrate liquid is dropped to between 55° and60° C. The pH of the solution is dropped from 6 to 6.5 to a rangebetween 3 and 5.5. Preferably, the pH of the solution is 4 to 4.5. Theglucoamylase is added to the solution and the reaction is carried outfor 24-72 hours, preferably 36-48 hours.

By improving the thermo stability of the Fungamyl-like alpha-amylasevariant according to the invention said alpha-amylases may be used forstarch liquefaction.

In an aspect the invention relates to the use of an alpha-amylasevariant of the invention in a starch conversion process.

Brewing

The alpha-amylase variant of the invention may also be used in brewingprocesses.

High Maltose Syrup Production (55% Maltose)

A variant of the invention may be used for maltose production. Highmaltose syrup is typically produced as follows:

Production of High Maltose Syrup (containing 50-55% maltose)

To produce “High Maltose Syrup” starch is liquefied to DE 10-20. The pHand temperature of the liquefied starch is adjusted to 65° C. and to apH around 5.0, respectively, and is subjected to maltogenicalpha-amylase activity (e.g., Bacillus stearothermophilus amylase, suchas Maltogenase™ 4000 L, 0.4 l/t DS (Novo Nordisk)), pullulanase activity(e.g., Bacillus pullulanase, such as Promozyme™ 600 L, 0.3 l/t DS (NovoNordisk)) and alpha-amylase activity (e.g., BAN 240 L or Termamyl™ 120L, type LS, 0.4 kg/t DS (Novo Nordisk)) for 24-41 hours. The specificprocess time depends on the desired saccharide spectrum to be achieved.By increasing the dosage of the maltogenic alpha-amylase and pullulanasethe maltose content can be increased.

Alternatively, “High Maltose Syrup” may be produced by first liquefyingstarch to DE 10-20 and then adjusting the pH and temperature to 55° C.and a pH around 5.5, respectively, and subjecting the liquefied starchto a fungal alpha-amylase activity (e.g., Bacillus stearothermophilusamylase, such as Fungamyl™ 800L (Novo Nordisk)) for 22-44 hours. Thedosage of fungal alpha-amylase depends on the saccharification timeforeseen, e.g., 200 g/t DS for 44 hours and 400 g/t DS for 22 hours.

To produce “High Maltose Syrup” starch with maltose content of 55-65%starch is liquefied to DE 10-20. The pH and temperature of the liquefiedstarch is adjusted to 60° C. and to a pH around 6, respectively, and issubjected to maltogenic alpha-amylase activity (e.g., Maltogenase™ 4000L, 0.25-1.0 l/t DS (Novo Nordisk)), and fungal alpha-amylase activity(e.g., Aspergillus amylase, such as Fungamyl™ 800 L, 0.4-1.0 kg/t DS(Novo Nordisk) for 24-48 hours.

Alternatively, the liquefied starch may adjusted to a temperature of 65°C. and a pH around 5.0 and subjected to maltogenic alpha-amylaseactivity (e.g., Bacillus stearothermophilus amylase, such asMaltogenase™ 4000 L, 0.5-1.0 l/t DS), and pullulanase activity (e.g.,Bacillus pullulanase, such as Promozyme™ 600 L, 0.5-1.0 l/t DS) for18-42 hours.

According to the invention one or more Fungamyl-like variants of theinvention may be used instead of or together with the above mentionedfungal alpha-amylase activity.

Baking

The alpha-amylase variant of the invention may also be used in bakingprocesses.

Use

In one aspect the invention relates to the used of a variant of theinvention for starch conversion, alcohol production, brewing, baking.

Processes of the Invention

The invention also relates to a process of producing maltose syrupcomprising the steps of:

-   1) liquefying starch in the presence of an alpha-amylase;-   2) dextrinization in the presence of a fungal alpha-amylase variant    of the invention; and-   3) recovery of the syrup; and optional purification of the syrup.

The alpha-amylase used for liquefaction in step 1) may be anyalpha-amylase. Preferred alpha-amylase are Bacillus alpha-amylases, suchas a Termamyl-like alpha-amylase, which including the B. licheniformisalpha-amylase (commercially available as Termamyl™ (Novo Nordisk)), theB. amyloliquefaciens alpha-amylase (sold as BAN (Novo Nordisk), the B.stearothermophilus alpha-amylase (sold as Termamyl™ 120 L type S), Thealpha-amylases derived from a strain of the Bacillus sp. NCIB 12289,NCIB 12512, NCIB 12513 or DSM 9375, all of which are described in detailin WO 95/26397, and the alpha-amylase described by Tsukamoto et al.,Biochemical and Biophysical Research Communications, 151 (1988), pp.25-31. Alpha-amylases within the definition of “Termamyl-likealpha-amylase” are defined in for instance WO 96/23874 (Novo Nordisk).

In another aspect the invention relates to a process of producingmaltose comprising the steps of:

-   1) liquefying starch at a temperature of 140-160° C. at a pH of 4-6;-   2) dextrinization at a temperature in the range from 60-95° C., in    particular at 65-85° C., such as 70-80° C., at a pH 4-6 in the    presence of a fungal alpha-amylase variant of the invention; and-   3) recovery of the syrup; and optional purification of the syrup.

In an embodiment of the invention an effective amount of glucoamylase isadded in step 2). The syrup will in this embodiment (including treatmentwith a glucoamylase) not be maltose syrup, but syrup with a differentsugar profile.

The glucoamylase may be an Aspergillus glucoamylase, in particular anAspergillus niger glucoamylase.

Alternatively, the process comprising the steps of:

-   1) liquefying starch at a temperature of 95-110° C. at a pH of 4-6    in the presence of a Bacillus alpha-amylase;-   2) liquefying at a temperature in the range from 70-95° C. at a pH    4-6 in the presence of a fungal alpha-amylase variant of the    invention, followed by recovery and/or optional purification of the    product obtained.    Immobilized Fungal Alpha-Amylase Variants of the Invention

In an aspect the invention relates to an immobilized alpha-amylasevariant of the invention. The alpha-amylase variant may be immobilizedusing any suitable method know in the art such as method used forglucose isomerase in U.S. Pat. No. 4,687,742.

MATERIALS AND METHODS

Material:

Enzymes:

FUNGAMYL®: fungal alpha-amylase derived from Aspergillus oryzae(available from Novo Nordisk) and shown in SEQ ID NO: 2.

Host cell:

A. oryzae JaL125: Aspergillus oryzae IFO 4177 available from Institutefor Fermention, Osaka; 17-25 Juso Hammachi 2-Chome Yodogawa-ku, Osaka,Japan, having the alkaline protease gene named “alp” (described byMurakami K et al., (1991), Agric. Biol. Chem. 55, p. 2807-2811) deletedby a one step gene replacement method (described by G. May in “AppliedMolecular Genetics of Filamentous Fungi” (1992), p. 1-25. Eds. J. R.Kinghorn and G. Turner; Blackie Academic and Professional), using the A.oryzae pyrG gene as marker. Strain JaL 125 is further disclosed in WO97/35956 (Novo Nordisk).

Micro-Organisms:

-   Strain: Saccharomyces cerevisiae YNG318: MATαleu2-Δ2 ura3-52    his4-539 pep4-Δ1[cir+]    Methods:    Transformation of Aspergillus oryzae (General Procedure)

100 ml of YPD (Sherman et al., (1981), Methods in Yeast Genetics, ColdSpring Harbor Laboratory) are inoculated with spores of A. oryzae andincubated with shaking for about 24 hours. The mycelium is harvested byfiltration through miracloth and washed with 200 ml of 0.6 M MgSO₄. Themycelium is suspended in 15 ml of 1.2 M MgSO₄, 10 mM NaH₂PO₄, pH 5.8.The suspension is cooled on ice and 1 ml of buffer containing 120 mg ofNovozym™ 234 is added. After 5 min., 1 ml of 12 mg/ml BSA (Sigma typeH25) is added and incubation with gentle agitation continued for 1.5-2.5hours at 37C until a large number of protoplasts is visible in a sampleinspected under the microscope.

The suspension is filtered through miracloth, the filtrate transferredto a sterile tube and overlayed with 5 ml of 0.6 M sorbitol, 100 mMTris-HCl, pH 7.0. Centrifugation is performed for 15 min. at 1000 g andthe protoplasts are collected from the top of the MgSO₄ cushion. 2volumes of STC (1.2 M sorbitol, 10 mM Tris-HCl, pH 7.5, 10 mM CaCl₂) areadded to the protoplast suspension and the mixture is centrifugated for5 min. at 1000 g. The protoplast pellet is resuspended in 3 ml of STCand repelleted. This is repeated. Finally, the protoplasts areresuspended in 0.2-1 ml of STC.

100 micro liter of protoplast suspension are mixed with 5-25 micro gramsof p3SR2 (an A. niulans amdS gene carrying plasmid described in Hynes etal., Mol. and Cel. Biol., Vol. 3, No. 8, 1430-1439, August 1983) in 10micro liter of STC. The mixture is left at room temperature for 25minutes 0.2 ml of 60% PEG 4000 (BDH 29576), 10 mM CaCl₂ and 10 mMTris-HCl, pH 7.5 is added and carefully mixed (twice) and finally 0.85ml of the same solution are added and carefully mixed. The mixture isleft at room temperature for 25 min., spun at 2.500 g for 15 min. andthe pellet is resuspended in 2 ml of 1.2M sorbitol. After one moresedimentation the protoplasts are spread on minimal plates (Cove,(1966), Biochem. Biophys. Acta 113, 51-56) containing 1.0 M sucrose, pH7.0, 10 mM acetamide as nitrogen source and 20 mM CsCl to inhibitbackground growth. After incubation for 4-7 days at 37C spores arepicked, suspended in sterile water and spread for single colonies. Thisprocedure is repeated and spores of a single colony after the secondre-isolation are stored as a defined transformant.

Fed Batch Fermentation

Fed batch fermentation is performed in a medium comprising maltodextrinas a carbon source, urea as a nitrogen source and yeast extract. The fedbatch fermentation is performed by inoculating a shake flask culture ofA. oryzae host cells in question into a medium comprising 3.5% of thecarbon source and 0.5% of the nitrogen source. After 24 hours ofcultivation at pH 5.0 and 34° C. the continuous supply of additionalcarbon and nitrogen sources are initiated. The carbon source is kept asthe limiting factor and it is secured that oxygen is present in excess.The fed batch cultivation is continued for 4 days, after which theenzymes can be recovered by centrifugation, ultrafiltration, clearfiltration and germ filtration. Further purification may be done byanion-exchange chromatographic methods known in the art.

Purification

The culture broth is filtrated and added ammonium sulphate (AMS) to aconcentration of 1.7 M AMS and pH is adjusted to pH 5. Precipitatedmaterial is removed by centrifugation on the solution containingalpha-amylase activity is applied on a Toyo Pearl Butyl columnpreviously equilibrated in 1.7 M AMS, 20 mM sodium acetate, pH 5.Unbound material is washed out with the equilibration buffer. Boundproteins are eluted with 10 mM sodium acetate, pH 4.5 using a lineargradient from 1.7-0 M AMS over 10 column volumes. Glucoamylasecontaining fractions are collected ad dialysed against 20 mM sodiumacetate, pH 4.5.

Screening for Thermostable Alpha-Amylase Variants

The libraries are screened in the thermostable filter assay describedbelow.

Filter Assay for Thermostability

Yeast libraries are plated on a sandwich of cellulose acetate (OE 67,Schleicher & Schuell, Dassel, Germany)—and nitrocellulose filters(Protran-Ba 85, Schleicher & Schuell, Dassel, Germany) on SC ura-agarplates with 100 micro gram/ml ampicillin at 30° C. for at least 72 hrs.The colonies are replica plated to PVDF filters (Immobilon-P, Millipore,Bedford) activated with methanol for 1 min and subsequently washed in0.1 M NaAc and then incubated at room temperature for 2 hours. Coloniesare washed from PVDF filters with tap water. Each filter sandwiches andPVDF filters are specifically marked with a needle before incubation inorder to be able to localise positive variants on the filters after thescreening. The PVDF filters with bound variants are transferred to acontainer with 0.1 M NaAc, pH 4.5 and incubated at 47° C. for 15minutes. The sandwich of cellulose acetate and nitrocellulose filters onSC ura-agar plates are stored at room temperature until use. Afterincubation, the residual activities are detected on plates containing 5%maltose, 1% agarose, 50 mM NaAc, pH 4.5. The assay plates with PVDFfilters are marked the same way as the filter sandwiches and incubatedfor 2 hrs. at 50° C. After removal of the PVDF filters, the assay platesare stained with Glucose GOD perid (Boehringer Mannheim GmbH, Germany).Variants with residual activity are detected on assay plates as darkgreen spots on white background. The improved variants are located onthe storage plates. Improved variants are re-screened twice under thesame conditions as the first screen.

Determination of FAU Activity

One Fungal Alpha-Amylase Unit (FAU) is defined as the amount of enzyme,which breaks down 5.26 g starch (Merck Amylum solubile Erg. B.6, Batch9947275) per hour at Novo Nordisk's standard method for determination ofalpha-amylase based upon the following standard conditions:

-   Substrate . . . Soluble starch-   Temperature . . . 37° C.-   PH . . . 4.7-   Reaction time . . . 7-20 minutes

A detailed description of Novo Nordisk's method is available on request.

Determination of Acid Alpha-Amylase Activity (AFAU)

Acid alpha-amylase activity is measured in AFAU (Acid FungalAlpha-amylase Units), which are determined relative to an enzymestandard.

The standard used is AMG 300 L (from Novo Nordiks). The neutralalpha-amylase in this AMG falls after storage at room temperature for 3weeks from approx. 1 FAU/mL to below 0.05 FAU/mL.

The acid alpha-amylase activity in this AMG standard is determined inaccordance with AF 9 1/3 (Novo method for the determination of fungalalpha-amylase). In this method, 1 FAU is defined as the amount ofenzyme, which degrades 5.260 mg starch dry matter per hour understandard conditions.

Iodine forms a blue complex with starch but not with its degradationproducts. The intensity of colour is therefore directly proportional tothe concentration of starch. Amylase activity is determined usingreverse colorimetry as a reduction in the concentration of starch underspecified analytic conditions.

-   Blue/violet t=23 sec. Decoloration    Standard Conditions/Reaction Conditions: (Per Minute)-   Substrate: starch, approx. 0.17 g/L-   Buffer: Citate, approx. 0.03 M-   Iodine (I₂): 0.03 g/L-   CaCl₂: 1.85 mM-   pH: 2.50 ±0.05-   Incubation temperature: 40° C.-   Reaction time: 23 seconds-   Wavelength: lambda=590 nm-   Enzyme concentration: 0.025 AFAU/mL-   Enzyme working range: 0.01-0.04 AFAU/mL

Further details can be found in EB-SM-0259.02/01 available on requestfrom Novo Nordisk, and incorporated by reference.

Thermal/pH Stability Determination of Variant of the Invention

The thermal stability of variants of the invention is tested using thefollowing method: 950 micro liter 0.1 M Citrate+4.3 mM Ca²⁺buffer isincubated for 1 hour at 60° C. 50 micro liter enzyme in buffer (4AFAU/ml) is added. 2×40 micro liter samples are taken at 0 and 60minutes and chilled on ice. The activity (AFAU/ml) measured beforeincubation (0 minutes) is used as reference (100%). The decline inpercent is calculated as a function of the incubation time.

To determine the Thermal stability the test is repeated using differenttemperatures, for instance 50, 60, 70, 80 and 90° C.

To determine the pH stability the test is repeated using different pHs,for instance, pH 2.5; 3; 3.5; 4; 4.5; 5.

General Method for Random Mutagenesis by Use of the DOPE Program

The random mutagenesis may be carried out as follows:

1. Select regions of interest for modification in the parent enzyme,

2. Decide on mutation sites and non-mutated sites in the selectedregion,

3. Decide on which kind of mutations should be carried out, e.g. withrespect to the desired stability and/or performance of the variant to beconstructed,

4. Select structurally reasonable mutations,

5. Adjust the residues selected by step 3 with regard to step 4.

6. Analyze by use of a suitable dope algorithm the nucleotidedistribution.

7. If necessary, adjust the wanted residues to genetic code realism,e.g., taking into account constraints resulting from the genetic code,e.g., in order to avoid introduction of stop codons; the skilled personwill be aware that some codon combinations cannot be used in practiceand will need to be adapted

8. Make primers

9. Perform random mutagenesis by use of the primers

10. Select resulting glucoamylase variants by screening for the desiredimproved properties.

Dope Algorithm

Suitable dope algorithms for use in step 6 are well known in the art.One such algorithm is described by Tomandl, D. et al., 1997, Journal ofComputer-Aided Molecular Design 11:29-38. Another algorithm is DOPE(Jensen, L J, Andersen, K V, Svendsen, A, and Kretzschmar, T (1998)Nucleic Acids Research 26:697-702).

EXAMPLES Example 1

Construction of variant Q153S

For the construction of variants of the TAKA-amylase enzyme (Fungamyl™shown in SEQ ID NOS: 1 and 2) the commercial kit, Chameleondouble-stranded, site-directed mutagenesis kit is used according to themanufacturer's instructions.

The gene encoding the amylase enzyme in question is in plasmid pTAKA17(EP 238,023, figure 2 and Example 2). In accordance with themanufacturer's instructions the ScaI site of the Ampicillin gene ofpTAKA17 is changed to a Mlul site by use of the following primer:

Primer 7258:

-   5′p gaa tga ctt ggt tga cgc gtc acc agt cac 3′ (SEQ ID NO: 3)

The ScaI site in an intron in the Amylase gene is removed using theprimer

Primer 1:

-   5′p ATG GTT CAT TTC AGA ACT GAC ATT GAG TAA (SEQ ID NO: 4)

The desired mutation is introduced into the amylase gene in question byaddition of an appropriate oligos comprising the desired mutation.

To introduce a mutation such as Q153S an oligo is design:

Primer 2:

-   5′P TTC TGT TTC ATT TCG AAC TAT GAA GAT (SEQ ID NO: 5)

The pTAKA17 vector comprising the amylase gene in question is then usedas a template for DNA polymerase, DNA ligase (for ligation to5′Phosphate (5′P) on the oligoes), and the oligoes 7258, primer 1 andprimer 2.

DNA-prep. are made, and the introduction of the mutation is verified bysequencing.

The DNA prep. is transformed in Aspergillus oryzae host cell as describein the “Materials & Methods” section and the transformants are screenedfor amylase activity.

Example 2

Increased Thermo Stability

The variant constructed in Example 1 is tested for increasedthermostability in accordance with the thermo stability determinationassay disclosed in the “Materials & Methods” section.

Example 3

Increased Acidic Stability

The variant constructed in Example 1 is tested for increased stabilityat acidic pH in accordance with the pH stability determination assaydisclosed in the “Materials & Methods” section.

1. A variant of a parent Fungamyl-like alpha-amylase, comprising analteration at one or more regions selected from the group consisting ofregion 98-110 and region 161-167, wherein (a) the alteration(s) areindependently (i) an insertion of an amino acid downstream of the aminoacid which occupies the position, (ii) a deletion of the amino acidwhich occupies the position, or (iii) a substitution of the amino acidwhich occupies the position with a different amino acid, (b) the varianthas alpha-amylase activity; (c) each region or position corresponds to aregion or position of the amino acid sequence of the parentFungamyl-like alpha-amylase having the amino acid sequence of SEQ ID NO:2 and (d) wherein the parent Fungamyl-like alpha-amylase is analpha-amylase from Aspergillus niger.
 2. The variant of claim 1, whereinthe variant further includes a substitution: Q153S.
 3. The variant ofclaim 1, wherein the variant has improved thermostability or increasedstability at acidic pH or improved thermostability and increasedstability at acidic pH.
 4. A composition for producing high maltosesyrup comprising the variant of claim
 1. 5. A dough improvingcomposition, comprising the variant of claim
 1. 6. A brewingcomposition, comprising the variant of claim
 1. 7. The brewingcomposition of claim 6, further comprising at least one additionalenzyme wherein the additional enzyme is a beta-amylase or an isoamylase.8. A composition for producing alcohol, comprising the variant ofclaim
 1. 9. The variant of claim 1 wherein the variant is immobilized.10. The variant of claim 1, wherein the alteration is an alteration inRegion 98-110.
 11. The variant of claim 1, wherein the alteration is analteration in Region 161-167.
 12. The variant of claim 1, wherein thevariant comprises an alteration at a position corresponding to position98 in SEQ ID NO:2.
 13. The variant of claim 1, wherein the variantcomprises an alteration at a position corresponding to position 99 inSEQ ID NO:2.
 14. The variant of claim 1, wherein the variant comprisesan alteration at a position corresponding to position 100 in SEQ IDNO:2.
 15. The variant of claim 1, wherein the variant comprises analteration at a position corresponding to position 101 in SEQ ID NO:2.16. The variant of claim 1, wherein the variant comprises an alterationat a position corresponding to position 102 in SEQ ID NO:2.
 17. Thevariant of claim 1, wherein the variant comprises an alteration at aposition corresponding to position 103 in SEQ ID NO:2.
 18. The variantof claim 1, wherein the variant comprises an alteration at a positioncorresponding to position 104 in SEQ ID NO:2.
 19. The variant of claim1, wherein the variant comprises an alteration at a positioncorresponding to position 105 in SEQ ID NO:2.
 20. The variant of claim1, wherein the variant comprises an alteration at a positioncorresponding to position 106 in SEQ ID NO:2.
 21. The variant of claim1, wherein the variant comprises an alteration at a positioncorresponding to position 107 in SEQ ID NO:2.
 22. The variant of claim1, wherein the variant comprises an alteration at a positioncorresponding to position 108 in SEQ ID NO:2.
 23. The variant of claim1, wherein the variant comprises an alteration at a positioncorresponding to position 109 in SEQ ID NO:2.
 24. The variant of claim1, wherein the variant comprises an alteration at a positioncorresponding to position 110 in SEQ ID NO:2.
 25. The variant of claim1, wherein the variant comprises an alteration at a positioncorresponding to position 161 in SEQ ID NO:2.
 26. The variant of claim1, wherein the variant comprises an alteration at a positioncorresponding to position 162 in SEQ ID NO:2.
 27. The variant of claim1, wherein the variant comprises an alteration at a positioncorresponding to position 163 in SEQ ID NO:2.
 28. The variant of claim1, wherein the variant comprises an alteration at a positioncorresponding to position 164 in SEQ ID NO:2.
 29. The variant of claim1, wherein the variant comprises an alteration at a positioncorresponding to position 165 in SEQ ID NO:2.
 30. The variant of claim1, wherein the variant comprises an alteration at a positioncorresponding to position 166 in SEQ ID NO:2.
 31. The variant of claim1, wherein the variant comprises an alteration at a positioncorresponding to position 167 in SEQ ID NO:2.